PROJECT SUMMARY Proteins fold into their 3-dimensional shape with the help of chaperones and other protein folding factors, which together comprise the proteostasis network (PN). During the protein quality control process, transient binding interactions between individual client proteins and proteostasis factors mediate folding into native functional structures, thereby ensuring trafficking to the correct cellular destination, or facilitating degradation of detrimental misfolded states. Consequently, imbalances in interactions between proteostasis factors and clients proteins result in quality control defects that lead to diverse protein misfolding diseases including highly prevalent neurodegenerative diseases such as Alzheimer?s and Parkinson?s Disease. The folding, maturation and trafficking of large multi-domain and multi-subunits proteins is a complex and highly client-specific process that depends on engaging the appropriate component of the PN at the correct time. Many proteostasis dependencies for individual client proteins, are known, but little is understood about the order of engagement, and whether correct sequential interactions are required for proper folding and trafficking. Understanding the coordination of the PN interaction with client proteins at an organelle- or cell-wide level will be crucial to determine the mechanisms of quality control defects that impact protein misfolding diseases. We utilize the power of chemical biology and quantitative proteomics tools to determine the interaction dynamics between disease-associated protein variants and the PN. This enables us to investigate the mechanism by which altered progression through the PN impacts protein quality control. To probe the progression of the client proteins through protein folding and trafficking pathways, we will establish new proteomics methodology to elucidate dynamically changing interactions both globally and in a time-dependent manner. We will examine the coordination requirement of different proteostasis pathways as they affect protein aggregation (thryroglobulin) and loss-of-function protein misfolding (cystic fibrosis transmembrance conductance regulator ? CFTR). Aggregation of destabilized thyroglobulin variants, a thyroid hormone precursor, is a leading cause for congenital hypothyroidism, while misfolding and pre-mature degradation of CFTR variants is the primary cause of Cystic Fibrosis. In particular, we will assess how protein quality control of mutant variants of these proteins is impacted by altered PN interactions and how established and new approaches to manipulate proteostasis pathways can correct these quality control defects. Results from our studies will provide significant new insights into how the dynamics of protein folding and trafficking pathways can be manipulated therapeutically for disease intervention.